Abstract Major depressive disorder (MDD) is a devastating mental illness arising from a combination of genetic, epigenetic, and environmental influences. Despite decades of investigation, our ability to diagnose and treat MDD remains limited, and a large fraction of MDD patients fail to respond to available treatment options. Epigenetic genome changes likely play a significant role in the pathophysiology of depression, especially since environmental stimuli and experience are important contributors to the development of MDD. We propose to couple technology development and circuit-specific epigenetic analysis of genome methylation and chromatin remodeling to reverse engineer the epigenetic mechanisms underlying major depressive disorder, and to discover novel drug targets for developing fundamentally new classes of antidepressants. Using animal models of depression we will identify specific circuits of cells in the brain whose functions are compromised in the disease state and determine the contributing epigenetic mechanisms. To enable the proposed research, we will develop an innovative platform of technologies to enable targeted genome and epigenome modifications and apply it to systematically identify epigenetic mechanisms in specific circuit components underlying depression. We will also explore the identified epigenetic mechanisms for developing new classes of antidepressants. In addition to our core technologies for genome and epigenome engineering, we will integrate a comprehensive range of technical expertise spanning electrophysiology, imaging, behavioral analysis, computational biology, synthetic biology, high-throughput genome and epigenome analysis, and highthroughput drug screening and assay development. The successful completion of our vision will yield four broad impacts: I. Pioneer a new approach for drug target discovery that has implications for a broad range of developmental and chronic illnesses. II. Develop a robust technology platform for large-scale targeted genomic engineering to enable more complete recapitulation of human disease genotypes in animal models. We will enable precise introduction of combinations of disease-associated genetic mutations into a single animal model. III. Develop a technology for targeted epigenome modification to enable direct functional testing of causal links between specific epigenetic modifications and disease pathophysiology. IV. Identify fundamentally new classes of therapeutics for major depression.